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Measurement of the content of limit-dextrinase in cereal flours.

Procedures for the quantitative extraction, activation, and assay of limit-dextrinase in cereal flours have been developed. Extraction and activation require incubation in buffer containing 20mm cysteine for at least 16 h or with 25mm dithiothreitol for 5 h. Activity is assayed with a soluble, dyed substrate (Red-Pullulan) or an insoluble, dyed, and cross-linked substrate (Azurine-CL-Pullulan) which is dispensed in tablet form (Limit-DextriZyme tablets).

A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis.

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85°C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80°C for 4 h and exhibited a half-life of 50 min at 85°C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed α-1,6 glycosidic linkages of pullulan, producing maltotriose, and also α-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.

Aims: The aim of this study was to determine the diversity of moderately halophilic bacteria with hydrolase activities. Methods and Results: Screening bacteria from different hypersaline environments in South Spain led to the isolation of a total of 122 moderately halophilic bacteria able to produce different hydrolases (amylases, DNases, lipases, proteases and pullulanases). These bacteria are able to grow optimally in media with 5–15% salts and in most cases up to 20–25% salts. In contrast to strains belonging to previously described species, that showed very little hydrolase activities, environmental isolates produced a great variety of hydrolases. These strains were identified as members of the genera: Salinivibrio (55 strains), Halomonas (25 strains), Chromohalobacter (two strains), Bacillus-Salibacillus (29 strains), Salinicoccus (two strains) and Marinococcus (one strain), as well as eight non-identified isolates. Conclusions: Moderately halophilic bacteria are a source of hydrolytic enzymes such as amylases, DNases, lipases, proteases and pullulanases. Significance and Impact of the Study: Although most culture collection strains are not able to produce hydrolases, it has been shown that environmental isolates can produce these potentially biotechnological important enzymes.

Analysis and characterization of cultivable extremophilic hydrolytic bacterial community in heavy‐metal‐contaminated soils from the Atacama Desert and their biotechnological potentials.

Aims: To isolate and characterize the cultivable community of hydrolase producers (amylase, protease, lipase, DNase, xylanase and pullulanase) inhabiting heavy-metal-contaminated soils in extreme conditions from the Atacama Desert. Methods and Results: A total of 25 bacterial strains showing hydrolytic activities have been selected including halotolerants, extremely halotolerants and moderate halophiles. Most hydrolase producers were assigned to the family Bacillaceae, belonging to the genera Bacillus (nine strains), Halobacillus (seven strains) and Thalassobacillus (five strains) and four isolates were related to members of the families Pseudomonadaceae, Halomonadaceae and Staphylococcaceae. The selected strains were then characterized for their tolerance pattern to six heavy metals, measured as minimal inhibitory concentrations (MICs). Conclusions: The diversity found in the cultivable bacterial community analysed is more limited than that detected in other ecological studies owing to the restrictive conditions used in the screening. The dominant bacteria were Firmicutes and particularly, species related to the genus Bacillus. Significance and Impact of the Study: This study is focused on the characterization of extremophilic hydrolytic bacteria, providing candidates as a source of novel enzymes with biotechnological applications.

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4°C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses α and γof Proteobacteria, the Bacillus–Clostridium group, the order Actinomycetales, and the Cytophaga–Flexibacter–Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria γ subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25°C. Only a few strains were psychrophilic, with an optimal growth at 10–15°C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4°C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, α-amylase and β-galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10°C, and almost no production was detected at higher temperatures (20–30°C). Catalytic activity was detected even below the freezing point of water (at −5°C), demonstrating the unique properties of these enzymes.

A number of bacteria were isolated from sea water in Skagerrak, Denmark, at 30 m depth. Two of the isolates, strains D28 and D30T, belonged to the Flavobacteriaceae within the Cytophaga–Flavobacterium–Bacteroides group. Sequencing of 16S rRNA genes of the two strains indicated strongly that they belonged to the genus Tenacibaculum and that they showed greatest similarity to the species Tenacibaculum amylolyticum and Tenacibaculum mesophilum. DNA–DNA hybridization values, DNA base composition and phenotypic characteristics separated the Skagerrak strains from the other species within Tenacibaculum. Thus, it is concluded that the strains belong to a novel species within the genus Tenacibaculum, for which the name Tenacibaculum skagerrakense sp. nov. is proposed, with strain D30T (=ATCC BAA-458T=DSM 14836T) as the type strain.

Purification and characterisation of two extremely halotolerant xylanases from a novel halophilic bacterium.

The present work reports for the first time the purification and characterisation of two extremely halotolerant endo-xylanases from a novel halophilic bacterium, strain CL8. Purification of the two xylanases, Xyl 1 and 2, was achieved by anion exchange and hydrophobic interaction chromatography. The enzymes had relative molecular masses of 43 kDa and 62 kDa and pI of 5.0 and 3.4 respectively. Stimulation of activity by Ca+2, Mn+2, Mg+2, Ba+2, Li+2, NaN3, and isopropanol was observed. The Km and Vmax values determined for Xyl 1 with 4-O-methyl-D-glucuronoxylan are 5 mg/ml and 125,000 nkat/mg respectively. The corresponding values for Xyl 2 were 1 mg/ml and 143,000 nkat/mg protein. Xylobiose and xylotriose were the major end products for both endoxylanases. The xylanases were stable at pH 4–11 showing pH optima around pH 6. Xyl 1 shows maximal activity at 60°C, Xyl 2 at 65°C (at 4 M NaCl). The xylanases showed high temperature stability with half-lives at 60°C of 97 min and 192 min respectively. Both xylanases showed optimal activity at 1 M NaCl, but substantial activity remained for both enzymes at 5 M NaCl.

The Gram-positive, alkali- and salt-tolerant marine bacterium strain P203T is described together with its closest phylogenetic neighbour, terrestrial isolate LMG 21005T. Strain P203T was isolated from material from the sponge Plakortis simplex that was obtained from the Sula-Ridge, Norwegian Sea. Strain LMG 21005T was an undescribed strain that was isolated from a church wall mural in Germany. Strains P203T and LMG 21005T were identified as novel alkalitolerant members of the Bacillus rRNA group 6 with a 16S rRNA gene sequence similarity of 99.5 %. The closest described neighbour, Bacillus gibsonii DSM 8722T, showed 99.0 % gene sequence similarity with P203T and 98.8 % similarity with strain LMG 21005T. Despite the high 16S rRNA gene sequence similarity, DNA–DNA cross-hybridization revealed only 25.8–34.1 % similarity amongst the three strains. The DNA G+C contents were 41.1 mol% for strain P203T and 39.6 mol% for strain LMG 21005T. Both strains grew well between pH 7 and pH 11. Strain P203T showed growth at moderate temperatures (from 4 to 30°C) and in the presence of up to 12 % (w/v) NaCl at pH 9.7, whereas strain LMG 21005T was not salt tolerant (up to 4 % NaCl) and no growth was observed at 4°C. The major fatty acids of strains P203T, LMG 21005T and the type strain of B. gibsonii were the saturated terminally methyl-branched compounds iso-C15 : 0 (19.8, 15.6 and 28.0 %, respectively) and anteiso-C15 : 0 (57.1, 48.6 and 45.2 %, respectively). Physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains P203T and LMG 21005T from the six related Bacillus species with validly published names and supported the proposal of two novel species, Bacillus plakortidis [type strain P203T (=DSM 19153T=NCIMB 14288T)] and Bacillus murimartini [type strain LMG 21005T (=NCIMB 14102T)].

Cloning, sequencing, and characterization of a heat-and alkali-stable type I pullulanase from Anaerobranca gottschalkii.

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.

A novel aerobic, psychrotolerant marine bacterium was isolated at 4°C from seawater samples collected from Spitzbergen in the Arctic. The strain was a polar-flagellated, Gram-negative bacterium that grew optimally at 10–15°C and pH 7–8 in media containing 2–3 % NaCl (w/v), using various carbohydrates and organic acids as substrates. The main fatty acid components included 16 : 0 (12.7 % of total fatty acids), straight-chain saturated fatty acid methyl ester (FAME) and 16 : 1ω7c (40.2 %) monounsaturated FAME. Phylogenetic analysis revealed a close relationship (99 % 16S rRNA gene sequence similarity) between the novel isolate and Pseudoalteromonas elyakovii KMM 162T and some other species of the genus Pseudoalteromonas. The DNA G+C content of the novel strain was 39 mol%. DNA–DNA hybridization showed only 47.6 % DNA–DNA relatedness with P. elyakovii KMM 162T, 44.2 % with Pseudoalteromonas distincta KMM 638T and 22.6 % with Pseudoalteromonas nigrifaciens NCIMB 8614T Based on phylogenetic and phenotypic characteristics, this isolate represents a novel species of the genus Pseudoalteromonas for which the name Pseudoalteromonas arctica is proposed; the type strain is A 37-1-2T (=LMG 23753T=DSM 18437T).

Mining Dictyoglomus turgidum for enzymatically active carbohydrases.

The genome of Dictyoglomus turgidum was sequenced and analyzed for carbohydrases. The broad range of carbohydrate substrate utilization is reflected in the high number of glycosyl hydrolases, 54, and the high percentage of CAZymes present in the genome, 3.09% of its total genes. Screening a random clone library generated from D. turgidum resulted in the discovery of five novel biomass-degrading enzymes with low homology to known molecules. Whole genome sequencing of the organism followed by bioinformatics-directed amplification of selected genes resulted in the recovery of seven additional novel enzyme molecules. Based on the analysis of the genome, D. turgidum does not appear to degrade cellulose using either conventional soluble enzymes or a cellulosomal degradation system. The types and quantities of glycosyl hydrolases and carbohydrate-binding modules present in the genome suggest that D. turgidum degrades cellulose via a mechanism similar to that used by Cytophaga hutchinsonii and Fibrobacter succinogenes.

Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter.

Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.